Mucociliary functions are the first defense mechanism against various inhaled pollutants in conducting airway epithelium. Vitamin A is one of the most important factors involved in the regulation of these functions. Many studies have been carried out, but the nature of vitamin A regulation in these differentiated functions is still largely unclear. Studies with support from this grant (RO1-HL35635) have demonstrated that epithelial cells from human, monkey, and hamster tracheobronchial tissues are able to express mucous cell differentiation in culture and the differentiation is dependent on the presence of vitamin A. Without vitamin A, epithelial cells express squamous cell differentiation in culture. This competitive renewal application is concerned with the biology of mucous cell differentiation. Based on preliminary studies with the microinjection and genetic cloning, we hypothesize that vitamin A regulates mucous cell differentiation in two different stages. The first stage is a competent phase in which squamous epithelial cells become uncommitted to squamous or mucous cell differentiation after the vitamin A treatment. The second stage regards as a progressive phase in which uncommitted cells are committed to mucous cell differentiation after the vitamin A treatment. We also hypothesize that each stage involves different sets of new gene expression. It is proposed to investigate the gene expression in these two stages and to define the roles of these new genes in the regulation of mucous cell differentiation. Our specific aims are: 1) to clone and to characterize vitamin A-responsive genes with a focus on those involved in the early events and differentiation, 2) to define the level (i.e. transcription, translation) of gene expression regulated by vitamin A, 3) to determine whether these genes are necessary and sufficient for mucous cell differentiation, 4) to determine precursor-mucous cells relationship and cell type-specific gene expression in a biphasic organotypic culture system. Several vitamin A-responsive cDNA clones have been isolated and they will be used initially to define the stage of mucous cell differentiation and to elucidate the mechanism of vitamin A regulation.